Tryptophanyl-tRNA synthetase derived polypeptides useful for the regulation of angiongenesis
A technology of angiogenesis and new blood vessels, applied in sugar derivatives, cardiovascular system diseases, recombinant DNA technology, etc., can solve problems such as not easy to spread freely
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[0064] "Isolated" or "purified" when referring to a preparation prepared from a biological cell or host means any extract of cells containing the DNA or protein in question, including crude extracts of the DNA or protein of interest. For example, in the case of proteins, purified preparations in which the DNA or protein of interest may be present in various purities can be obtained according to individual techniques or a series of preparative or biochemical techniques. These procedures can include, for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation, and electrophoresis.
[0065] A "substantially pure" or "isolated" DNA or protein preparation means a preparation free of naturally occurring materials with which the DNA or protein is ordinarily associated in its natural state. "Essentially pure" should be understood to mean a "highly" purified preparation, which...
Embodiment 1
[0157] Example 1. Preparation of endotoxin-free recombinant TrpRS
[0158] Endotoxin-free recombinant human TrpRS was prepared as follows. Preparations encoding full-length TrpRS (amino acid residues 1-471 of SEQ ID NO: 1) or consisting essentially of residues 94-471 of SEQ ID NO: 1 (i.e., residues 94-471 of full-length TrpRS), called T2 truncated TrpRS (SEQ ID NO: 12), and a plasmid consisting essentially of residues 71-471 of SEQ ID NO: 1 , hereinafter referred to as T1 truncated TrpRS (SEQ ID NO: 13). Each plasmid also encoded a C-terminal tag containing six histidine residues (eg, amino acid residues 472-484 of SEQ ID NO: 1) and an initial methionine residue. Contains His 6 The tagged T1 has the amino acid sequence of SEQID NO: 5, and contains His 6 Tagged T2 has the amino acid sequence of SEQ ID NO:7.
[0159] The above plasmids were introduced into E. coli strain BL21(DE3) (Novagen, Madison, WI). A human mature EMAPII tagged with 6 histidine residues at the C-term...
Embodiment 2
[0161] Example 2. Cleavage of human TrpRS with PMN elastase
[0162]Cleavage of human TrpRS with PMN elastase was examined. TrpRS was treated with PMN in PBS (pH 7.4) for 0, 15, 30 or 60 minutes at a protease:protein ratio of 1:3000. After cleavage, samples were analyzed on a 12.5% SDS-polyacrylamide gel. Cleavage of the full-length TrpRS of approximately 53 kDa encoded by nucleotides 3428-4738 of DNA SEQ ID NO: 2 by PMN elastase yields a major fragment of approximately 46 kDa (SEQ ID NO: 5, T1 with a C-terminal histidine tag) and a minor fragment of approximately 43 kDa (SEQ ID NO: 7, T2 with a C-terminal histidine tag).
[0163] Carboxy-terminal His of the recombinant TrpRS protein 6 Western blot analysis with labeled antibodies revealed that both fragments have a carboxy-terminal His 6 mark. Therefore, only the amino termini of the two TrpRS fragments were truncated. The amino-terminal sequence of the TrpRS fragment was determined by Edman degradation using an ABI...
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